This invention relates to method and apparatus for increasing the throughput of analysis of compounds of a discrete size range.
The references mentioned herein are expressly incorporated by reference.
Separation of compounds is key to sample analysis in biology, chemistry, and physics. In numerous linear type (two-dimensional) separation systems, compounds are introduced into one end of a separation length and a flow force is then applied separating the compounds. Once the compounds are separated, the compounds can then be detected, analyzed and possibly isolated. Such separation systems include chromatography, electrophoresis, and centrifugal separation systems.
The very large number of compounds that need to be separated and analyzed has led to an increasing emphasis on high throughput systems. High throughput systems utilize automation, miniaturization, and parallel reactions or separations to increase the speed at which samples may be analyzed. This allows for an assay to maximize the information gained while minimizing the resources required to effect analysis. This achieves the benefit of lowering the amount of material required for each reaction, maximizing the value of costly analytical equipment, and efficiently using experimentalist time.
A number of fields require increased throughput systems. Environmental sampling, combinatorial chemistry, genomic assay and other fields require very large numbers of samples to be analyzed. High throughput analysis is also increasingly important to the analysis of nucleic acid sequences.
The development of the ability to generate very large numbers of discrete length oligonucleotide in samples has increased the demand for high throughput of oligonucleotide analytical systems. The polymerase chain reaction (PCR) (described in U.S. Pat. No. 4,683,202 to Mullis, K. et al.) discloses a method for amplification of nucleic acid sequences. In this method, high copy numbers of single strands of template nucleic acid sequences are generated. A short nucleic acid sequence primer is annealed to a specific sequence of the single stranded template nucleic acid. An initiator (DNA polymerase) adds nucleotide bases from solution to the 3xe2x80x2-end of the primer. By using matched sets of two primers, oligonucleotide products of specific known length may be amplified. In addition to PCR, use of restriction enzymes to cut nucleic acid strands into fragments of discrete lengths is used to detect specific sequence differences in DNA.
An additional need for high throughput nucleic acid analytical systems results from the very large size and variability of the genomes of various organisms. For example, human genome contains over three billion nucleotide base pairs. These three billion bases represent many thousands of genes. When analyzed on the base pair level, millions of single base variations exist in the human population. High throughput is required for systems used to analyze genomic variation of an organism or to track segregation within a genome.
Various methods can be used to detect genetic variability. These include restriction fragment length polymorphism (RFLP), analysis of short tandem repeat (STR) loci, and analysis of single nucleotide polymorphism.
RFLP is an assay for variation in DNA sequences at a site at which a particular restriction enzyme cuts. DNA variants will give different sizes of DNA fragments after digestion with a specific restriction enzyme. The use of probes to detect known sequences allows determination of restriction site variability proximate to a specific locus. RFLP can be combined with PCR to produce oligonucleotides of a discrete size range. In xe2x80x9cThe use of capillary electrophoresis for point mutation screeningxe2x80x9d in Trends Biotechnol., November 1997; 15(11):448-51 by Mitchelson, K.; Cheng, J.; Kricka, L., the authors described the widely used PCR-RFLP technique wherein the PCR reaction is run to produce amplified DNA fragments from a target template DNA. The amplified product is cleaved by a restriction endonuclease at specific cleavage sites to which the endonuclease binds and the size of resulting fragments determined. Presence or absence of a cleavage site is indicative of detection of the mutation of interest.
Short tandem repeat loci (variation in the number of short, tandem repeat units at a locus causing DNA length variability between alleles at that locus) may be used to assay DNA variability. The highly variable copy number of the short repeats serves as a genetic marker. Distinct polymorphic short tandem repeat (STR) loci are amplified and analyzed. U.S. Pat. No. 5,843,660 to Schumm et al. describes a procedure for using PCR to amplify STR loci. The amplification procedure produces a set of nucleic acid fragments within a discrete size range. The nucleic acid sequences of the discrete size range may then be separated and analyzed. In addition to RFLP and STR, single nucleotide polymorphisms may be used as markers of the genetic or phenotypic variability and as genetic markers. The use of single nucleotide polymorphism may present various advantages over either RFLP or STR analysis.
Numerous advantages are presented by single nucleotide polymorphism (SNP) analysis. Generally SNP analysis protocols produce small nucleic acid sequences of a discrete size range. SNP analysis may be rapid and adaptable to automation. This results in a reduction of the amount of reagents and other materials that is needed to effect this analysis. SNP genotyping is highly adaptable to high throughput systems. In the human genome, millions of individual single nucleotide polymorphisms exist. In contrast, much lower frequency of STR or RFLP variants occur.
The advantages of SNP analysis have made this analysis a preferred genomic marker assay. SNP analysis is useful for genotyping or enabling mutation scoring, genetic mapping, pharmacogenetic typing, phylogeny typing, and is adaptable to forensic and identity analysis. Single nucleotide polymorphisms may correlate to phenotypic expression and some SNP variants could serve as markers for disease states enabling simplified diagnostics. A relatively large number of SNP variants exist in the human genome, numbering in the millions. At specific loci, the variation may be between two nucleotides: a common-type variant nucleotide (common variant allele) and a rare-type variant nucleotide (rare variant allele). This binary variation (diallelic) enables allele analysis in which a variant may be categorized as either primary type or variant type. Thus unlike STR variation, SNP variation more rapidly may yield binary allele calls. RFLP variants are binary but cumbersome and expensive.
A number of techniques have been described to use single nucleotide polymorphism in genetic analysis. One such procedure is single nucleotide primer extension (SNuPE). An example is seen in U.S. Pat. No. 5,888,819 to Goelet et al. which describes the use of a PCR-like analysis system to gain information on genetic variation. In this procedure a single stranded template nucleic acid sequence is obtained and a primer is hybridized onto a conforming template sequence to form a template-primer duplex. A DNA polymerase extends the nucleic acid-primer duplex by one nucleotide. This can be enabled by using a terminator, such as a radio-labeled or fluorescence-labeled dideoxynucleotide, to ensure that only a single base is attached to the 3xe2x80x2-end of the primer. This protocol extends the primer by one base. Determination of which type of nucleotide was incorporated onto the 3xe2x80x2-end of the primer is enabled by the presence of the type of the detectable label.
U.S. Pat. No. 5,846,710 to Bajaj describes similar technology for single nucleotide polymorphism analysis. A single stranded sample oligonucleotide is mixed with a labeled nucleotide, a primer having a sequence complementary to a known sequence on the template oligonucleotide and an agent to induce DNA extension (such as a DNA polymerase). Other than the labeled nucleotide, no other nucleotides are included in the mixture. The mixture is then subjected to conditions under which the primer anneals to the sample single stranded oligonucleotide. The DNA polymerase then incorporates the labeled nucleotide if a base complementary to the labeled nucleotide is present at the location on the sample oligonucleotide at the single base opposite the base immediately upstream of the 3xe2x80x2-end of the primer. An article entitled xe2x80x9cCapillary Electrophoresis for the Detection of Known Point Mutations by Single-Nucleotide Primer Extension and Laser-Induced Fluorescence Detectionxe2x80x9d by Piggee, C. et al. in Journal of Chromatography A 781 (1997), p. 367-375 describes the separation of DNA fragments in a capillary electrophoresis format in conjunction with laser-induced fluorescence (LIF) detection. LIF was used to detect single nucleotide polymorphism using a method of single-nucleotide primer extension described in the previous two references. The labeled terminator was labeled with fluorescent dyes.
A number of different methods of analyzing single nucleotide polymorphisms by using single nucleotide primer extension reactions with detection of the resultant reaction products have been described. In Human Genetics (1999) 104:89-93, author Hoogendoorn, B. et al. in an article entitled xe2x80x9cGenotyping Single Nucleotide Polymorphism by Primer Extension and High Performance Liquid Chromatographyxe2x80x9d describes SNuPE reaction product analysis using high performance liquid chromatography (HPLC). This reference describes a xe2x80x9cminisequencingxe2x80x9d type analysis wherein a DNA sequence containing a single nucleotide polymorphism is amplified by the extension of an oligonucleotide primer which is annealed adjacent to the polymorphism. In the presence of a labeled terminator, the primer is extended by at least one base. In the PCR reaction mixture is included at least three of the four nucleotides and one terminator nucleotide. A DNA polymerase will add nucleotides to the 3xe2x80x2-end of the primer to a location on the sample DNA complementary to the terminator base. HPLC analysis of the primer extension reaction products from such a reaction results in chromatographic data from which the genotype of the polymorphic site, with genotype cells as a wild type or mutant type.
U.S. Pat. No. 5,885,775 to Haff et al. describes methods for single nucleotide polymorphism analysis by mass spectrometry. As in the previous methods, a PCR reaction including a nucleotide terminator generates a primer extension product of a discrete size range. The base composition of the extended primer is determined by mass spectrometry. The different weights of the various nucleoside bases allow the bases to be distinguished.
In many of these DNA analytical methods, a large number of oligonucleotides of a discrete size range are generated and analyzed. Various techniques may be adapted to increase the throughput of the analysis of the compounds. The use of high throughput techniques allows for the analysis of nucleic acid sequences at a faster rate.
Various techniques have been adapted to increase the throughput of the analysis of small compounds (e.g. nucleic acid sequences in a discrete size range). In Proceedings of the National Academy of Sciences, vol. 95, pages 2256-2261, March 1998 in an article entitled xe2x80x9cHigh-throughput genetic analysis using microfabricated 96-sample capillary electrophoresis microplatesxe2x80x9d by Simpson, P. et al., the authors disclose a capillary array electrophoresis system that can rapidly analyze 96 samples in a parallel electrophoresis procedure. This system utilizes a 96-sample well glass sandwich microplate with 48 separation channels. Two machined capillaries flow into each channel. Samples are injected into two ports on two capillary lengths which lead to a central detector. This permits the serial analysis of two different samples in each associated capillary with detection by a central detector. This technique requires the specialized microplate sample well with injection unit to produce the increased throughput. Multiple injection reservoirs and capillaries are required for the serial injection of two samples to be read by one detector.
Additional methods to increase throughput of genomic analysis are described in Human Molecular Genetics, volume 1, no. 6, page 391-395 in an article entitled xe2x80x9cRapid and Simultaneous Detection of Multiple Mutations by Pooled and Multiplexed Single Nucleotide Primer Extension: Application of the Study of Insulin-Responsive Glucose Transporter and Insulin Receptor Mutations in Non-Insulin-Dependent Diabetesxe2x80x9d by Krook, A. et al. In this publication, the authors describe two techniques employed in single nucleotide primer extension reactions that allow greater throughput by simultaneously examining multiple alleles at multiple loci.
The first technique described is the pooling of samples. By adding together a large number of samples prior to performing PCR reactions for a single locus allows for analysis of a large amount of samples in a single nucleotide primer extension reaction. This technique is most useful for the analysis of rare type alleles. If an analysis of the pool indicates the presence of a target sequence within the pool of samples, individual samples may be later analyzed to determine the individual containing the sequence of interest.
The second adaptation to increase throughput is the use of multiple primers of varied length during primer extension. Each primer is designed to bind to a specific known sequence with a polymorphic genetic variant positioned at the base located immediately downstream opposite the 3xe2x80x2-end of the primer. Using several primers of distinguishably different lengths allows throughput to be increased. A similar technique of increasing throughput is described in Nature Biotechnology, Vol. 16, December 1998 in an article entitled xe2x80x9cHigh Level Multiplex Genotyping by MAODI-TOF Mass Spectrometryxe2x80x9d by Ross, E. et al. This reference describes the use of a 12-primer system to analyze single nucleotide polymorphism at 12 different loci. The twelve primers are combined in a single PCR reaction. The reaction products are analyzed by mass spectrometry. The primers are of sufficiently different length to allow them to be distinguished by mass spectrometry.
An additional method to increase throughput is by the use of multiple dye labels. Attaching a dye having a specific characteristic excitation and emission wavelength to each nucleotide terminator used in a SNuPE reaction allows the simultaneous analysis of nucleotide variation. An optical detector can simultaneously distinguish the various wavelengths of each dye thereby detecting the specific nucleotide incorporated in the analyzed oligonucleotide.
It is the object of the invention to describe a technique and apparatus for further increasing the throughput of analytical systems designed to analyze small compounds, particularly DNA sequences of a discrete size range.
It is a further object to effect this increase in throughput with limited alteration to existing apparatus.
The above objects have been achieved through a method and apparatus in which injections into a separation length are spaced in time, with this spacing designed such that the separate injections are detectably separated at a detection location along the separation length distal from the sample injection location. The timing of the injection is designed such that when samples reach the detector, the samples are detectably separated and the compounds within each sample are likewise detectably separated.
As applied to capillary electrophoresis providing an array of capillaries and filling each capillary in the array with a separation media effects this method. The separation media may include a sieving matrix or may be a free solution capillary electrophoresis media. The method further includes preparing a plurality of samples wherein a percentage of the plurality of samples contains a target molecule of a discrete size range. Each different target molecule is labeled with a characteristic detectable labeling agent. The plurality of samples may be prepared or amplified in a number of ways including utilizing the polymerase chain reaction to produce oligonucleotides of a distinctive size range. In effecting the polymerase chain reaction, the use of multiple primers, pooling of samples, and multiple optically detectable labeling agents allows for an increase in throughput of sample analysis. The step of preparing a plurality of samples may include using single nucleotide primer extension to assay single nucleotide polymorphism at one or more loci in each separate sample preparation.
After the samples are prepared or amplified, they are transferred into a plurality of sample receptacle containers. Each sample receptacle container has a plurality of sample holding receptacles for holding the sample. An example of a sample receptacle container is a 96-well microplate. Each capillary in the capillary array is brought into contact with one sample in the sample receptacle container. A discrete selected amount of the sample may then be loaded into each capillary by an injector.
The samples are loaded in a batch, each batch representing the samples from a single sample receptacle container. The samples are then electrophoretically separated. The electrophoretic separation causes the samples to migrate away from the injector and towards a detector. After the samples have migrated a selected distance away from the injector, the injector injects another sample into each capillary in the array of capillaries from another sample receptacle container. The cycle of injection and separation is repeated a number of times. The array of capillaries is scanned at a detection region to detect the labels on the small target molecules that have been separated. This produces detection data that may then be analyzed.
The method uses the additional step of the inclusion with the samples of a quality metric standard. Such a standard could provide information on the efficacy of the sample separation, the functioning of the sample separation system, or the size of the compound being analyzed.
An alternative aspect of the invention is an apparatus for the ultra high throughput analysis of small compounds. The apparatus includes a capillary array with each capillary of the array filled with a separation media. Electrodes may be brought into electrical communication with the opposing ends of each capillary introducing electrical flow through the capillary. An injector is configured to inject samples from a multiwell sample plate into an injection end of each of the capillaries in the array. Associated with the injector is a timer that periodically instructs the injector to inject additional samples from additional multiwell plates into the capillaries of the capillary array. The capillary array has a detection area associated with the capillaries of the capillary array at an end distal from the injection end. A laser in the apparatus directs coherent light at the detection area that excites fluorescence from labeled samples within the capillaries. A multi-channel optical detector is positioned to detect this excited fluorescence.
The timer that is included with the apparatus may also be associated with the electrodes such that before the timer instructs the injector to inject additional samples into the capillary array, the timer first instructs the electrodes to pause the current flow through the capillary array. This timing system may be a circuit, a timing algorithm or some other timing means.
The apparatus may also include a multiwell sample plate transporter such as a conveyor or robotic arm to bring additional multiwell sample plates to the injector and a magazine for feeding the multiwell sample plates to the transporter.